A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette and a CDROM.
1. Field of the Invention
The present invention is directed towards the screening of different pig breeding lines by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CD 151. Selection of lines which are genetically low in or do not possess CD 151 produces offspring which are less susceptible or not susceptible at all to infection by porcine reproductive and respiratory syndrome virus (PRRSV). The present invention also concerns non-invasive methods for screening live pigs and swine germplasm for susceptibility to PRRSV. Additionally, the invention describes the development of cell lines for propagating high titer stocks for making killed and modified live virus vaccines in non-simian cell lines. Moreover, the invention describes a novel plasmid, useful in transforming previously non-susceptible cell lines into susceptible ones for producing the titer stocks used in vaccines. The invention also pertains to providing a tool for discovery of a novel class of anti-viral compounds that will block the interaction between CD 151 and PRRSV 3xe2x80x2-UTR RNA. This novel class of compounds has been termed anti-RNA Entry Proteins (anti-REPs).
2. Description of the Prior Art
Porcine reproductive and respiratory syndrome (PRRS) is a RNA virus which emerged in the late 1980""s as an important viral disease of swine. PRRS is the most important swine health problem today. The origin and evolution of PRRSV is not known. Thus, it is a novel emerging virus of pigs. This disease, which has previously been referred to as xe2x80x9cmystery swine diseasexe2x80x9d, xe2x80x9cswine infertility and respiratory syndromexe2x80x9d, or xe2x80x9cblue ear disease,xe2x80x9d is causing heavy losses in breeding herds of the United States and Canada. A similar disease has also appeared in much of Europe and the virus has been detected worldwide. The disease is manifested in two forms, one causing severe reproductive failure in pregnant sows, manifested in the form of premature farrowings, increased numbers of stillborn, mummified and weak-born pigs, decreased farrowing rate, and delayed return to estrus and the other producing respiratory distress in pigs evidenced by lesions that appear in the lungs of infected swine.
The reproductive form of the disease is described by Keffaber, K.K., xe2x80x9cReproductive Failure of Unknown Etiologyxe2x80x9d, American Association of Swine Practitioners Newsletter, 1:109 (1989). The most prominent clinical symptoms of the reproductive form of the disease are spontaneous late-term abortions, premature births (which can be as high as 20-30% of all births) and the farrowing of mummified fetuses, stillborn or sickly piglets. Such clinical symptoms will typically be observed in a herd from 4-16 weeks, or even longer. Stillborn fetuses in affected litters often are in the early stages of mummification, as evidenced by tan-brown discoloration of the skin and post-mortem autolysis. Dome-shaped malformations of fetal skulls is also sometimes seen. The infection of sows may go unnoticed, or may manifest itself by an impaired general condition lasting up to a few days. For example, the sows may go off feed, and experience body temperatures either above or below normal. In the farrowing phase, the sows may exhibit depression, lethargy, pyrexia and occasional vomiting. In some affected herds, up to 75% of all piglets may be lost. The economic consequences of the disease, accordingly, are devastating.
The respiratory form of the disease exhibits clinical signs which are most pronounced in piglets of 3-8 weeks in age, but are reported to occur in pigs of all ages in infected herds. The diseased piglets grow slowly, have roughened hair coats, respiratory distress (xe2x80x9cthumpingxe2x80x9d) and increased mortality (up to about 80% pre-weaning mortality). To combat the problems associated with infection by PRRSV, vaccines have been developed in an attempt to confer immunity to the current PRRSV strains. The present vaccines are only marginally effective and are all produced in cell lines of simian origin which possessed a risk of continuous introduction of primate viruses in swine populations. Findings in preliminary studies of gross and microscopic lesions of piglets affected with the respiratory form of the disease suggest that microscopic lung lesions are an important clinical feature of this disease.
PRRSV belongs within the order Nidovirales in the family Arteriviridae. Other members in the family include equine arteritis virus, lactate dehydrogenase elevating virus, and simian hemorrhagic fever virus. Currently, there is no known human arterivirus. The PRRSV genome is a positive sense RNA about 15.1 kb in length. Untranslated regions (UTR""s) of 156-220 nucleotides at the 5xe2x80x2 end and 59-117 nucleotides at the 3xe2x80x2 end flank the viral genome. The viral genome has eight overlapping open reading frames encoding functional and structural proteins. PRRSV grows primarily in the macrophages of infected pigs. In cell culture, the virus is known to grow in CL 2621, MA-104, MARC cell lines and in primary cultures of porcine alveolar macrophages. All of these continuous cell lines are of simian origin and pose a risk of introduction of primate viruses into swine populations. Entry of the virus occurs by receptor-mediated endocytosis and the receptor has been characterized as a heparin-like molecule. However, the mechanisms of how viral RNAs enter after endocytosis into the cell cytoplasm are not known.
Replication of arteriviruses is similar to that of the coronaviruses. Genomic and subgenomic (xe2x88x92) sense mRNAs are formed in the infected cells along with the (+) sense mRNAs. Subgenomic (xe2x88x92) sense mRNAs have been shown to function as the principal templates for mRNA synthesis in coronaviruses. Discontinuous transcription occurs in arteriviruses with the formation of a functionally monocistronic, 3xe2x80x2-coterminal, nested set of mRNAs. The common leader is joined to the coding region by consensus intergenic sequences through the junction sequence UCAACC. In mouse hepatitis virus (MHV), a corona virus, interaction of the leader, intergenic sequence and the body of the RNA involves cis and trans acting elements. The 3xe2x80x2UTR""s of MHV, coxsackie-, rhino- and polioviruses were shown to be essential for the transcription of the genome. There is little sequence complementarity between the 3xe2x80x2UTR and upstream regulatory sequences for interaction, therefore it may be mediated through RNA-protein-RNA interactions involving the viral or cellular proteins.
There are numerous studies on the interactions of viral and cellular proteins with 5xe2x80x2 and 3xe2x80x2 UTR""s of viruses. Sindbis virus, brome mosaic virus, QB phage and polioviruses require the interaction of certain host cell proteins with viral UTR""s for transcription to proceed. Although numerous proteins have been shown to bind to these regulatory regions, only a few of them have been characterized. La protein, poly (rC) binding protein 2, and polypyrimidine tract binding protein are shown to bind to UTR""s of poliovirus. Polypyrimidine tract binding protein and heterogeneous nuclear ribonucleoprotein have been shown to bind to the leader sequence of MHV. These proteins are predicted to play a role in mRNA splicing and transportation. Some cytoskeletal and chaperone proteins, like actin, tubulin, and heat shock proteins, are shown to have RNA binding activity. These proteins might play a structural role in viral RNA synthesis or in orienting and transporting the RNA replication complexes to the site of replication.
The study of host cell proteins that interact with viral RNAs is still in the infancy stage and there is a lack of important information regarding this interaction, especially with respect to host-susceptibility factors. Even in arteriviruses, such as PRRSV, the host susceptibility factors have not been studied. Thus, the markers for swine breeding for increased host susceptibility to PRRSV are not known. However, it is known that different breeds of pigs do differ in PRRSV susceptibility based on experimental infection followed by sacrificing the animals followed by further examination with histopathology and immunohistochemistry for interstitial pneumonia and presence of PRRSV antigen in the lungs.
Moreover, only simian cell lines provide the cell culture for current PRRSV vaccines which is a dangerous activity. The use of simian cell lines for these cell cultures might accidentally introduce primate viruses of significance into swine lines intended for xenotransplant purposes. Because swine are being increasingly explored as a source of xenotransplanted organs to meet the shortage of organ transplants for humans, the introduction of primate cell lines to swine populations may ultimately pose a risk to humans having xenotransplanted organs. Thus it would be prudent to avoid the use of simian cell lines in swine vaccine preparations.
Accordingly, what is needed in the art is a method of screening swine for susceptibility to PRRSV infection. Preferably, this screening should be non-invasive and able to be performed on a number of different animal fluids and cellular material, including swine germplasm and whole blood. Additionally what is needed is a method of using these screening results in a breeding program designed to lessen the susceptibility of offspring to PRRSV infection. What is further needed is a non-simian cell line for propagating high-titer PRRSV vaccine stock to avoid crossover of primate viruses into the swine population. Such a cell line will be especially adapted for vaccines used with xenotransplanted swine. Finally, what is needed is a class of compounds which can block the entry of viral RNA into cells.
The present invention solves the prior art problems mentioned above and provides a distinct advance in the state of the art. In particular, through the present invention, methods are provided which allow non-invasive genetic testing for PRRSV susceptibility, selection of swine for further breeding, the development of high-titer vaccines in non-simian cell lines, the development of compounds which can be used for PRRSV treatment, and the detection and characterization of the interaction between the 3xe2x80x2UTR RNA of the PRRS genome and susceptible cell lines, thereby leading to the development of novel anti-viral compounds called anti-RNA Entry Compounds.
Accordingly, one aspect of the present invention provides a method of screening swine using relatively non-invasive methods. For example, a blood sample could be drawn and the presence of CD 151 in platelets could be detected using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The knowledge gained from this type of screen could greatly improve swine breeding plans as swine with low levels or even no detectible CD 151 could be selected for further breeding. One example of a swine breeding program which utilizes advantages of the present invention compares the CD 151 levels of an individual swine to a known standard of CD 151. This known standard is generally found by comparing the CD 151 levels from a large number of samples of cellular material (e.g. fluid or tissue) from swine and represents the average level of CD 151 detected in the samples. As different cellular materials from the same animal may have different levels of CD 151, it is preferable to compare CD 151 levels from a cellular material of known origin (blood, blood platelets, sperm cells, germplasm, semen, ova, etc.) with the known standard for that specific cellular material. Moreover, as the known standard represents an average CD 151 level for swine, an individual swine may be selected for further breeding based on their detected CD 151 level in comparison with the average. Generally, it is preferable to select swine with CD 151 levels which are lower than average as this would tend to produce offspring with increased resistance to PRRSV infection. In this manner, swine having certain levels of CD 151 which place the swine into a particular desired percentile for CD 151 levels and which retain other desirable breeding characteristics would be selected for this further breeding. Preferably, these swine would rank in the lower 50th percentile for CD 151 levels. More preferably, these swine would rank in the lower 80th percentile for CD 151 levels, still more preferably, these swine would rank in the lower 90th percentile for CD 151 levels, and most preferably, these swine would rank in the lower 95th percentile for CD 151 levels. In other words, for this most preferred grouping, 95% of all swine would have higher CD 151 levels than the individual swine being tested. Of course, CD 151 deficient swine could be further bred to produce a xe2x80x9cknockoutxe2x80x9d line of swine which would be resistant to PRRSV infection.
In a related aspect of the present invention, cellular material in the form of germplasm such as sperm cells, or extended porcine semen could be screened for the presence of CD 151 prior to distribution to breeding facilities. Again, using the same general procedures described above, the knowledge gained from such a screen would be invaluable to future breeding plans and use of large amounts of germplasm. The ejaculation volume in swine is approximately 500 mL and the semen gets further extended so even if the animal is no longer on the farm or has died, or has been sold, the germplasm from a valuable boar can be used for many years and for many sows and could provide several future generations of piglets of desirable genotype or genetic makeup. Such frozen germplasm can also be screened for CD 151 levels and PRRSV susceptibility before artificial insemination. This screening could also indicate the susceptibility of animals or their offspring to infection by PRRSV as higher levels of CD 151 are correlated with higher virus production and release by cells. Thus, these tests will also be applicable for screening live animals on farms for their susceptibility to PRRSV. The selection of swine for further breeding based on CD 151 levels detected in germplasm would proceed as follows. A sample of germplasm would be taken and the amount or level of CD 151 in that sample would be determined by RT-PCR. This result (the amount of CD 151) would be compared with known standards for that sample as different types of germplasm would typically contain different amounts of CD 151. The known standards would be determined by the average of the quantitation of a large number of such samples. Swine exhibiting reduced levels of CD 151 in comparison to the standards (and thereby having a reduced susceptibility to PRRSV infection) yet still possessing other valuable breeding traits would be selected for further breeding. Preferably, these swine would have CD 151 levels lower than about 50% of all swine tested. More preferably, these levels will be lower than at least about 80%, still more preferably, at least about 90%, and still more preferably, at least about 95% of all swine tested. In other words, the CD 151 levels in the samples from these swine would be ranked approximately in the bottom 20%, 10%, or 5% of all swine tested. Of course, many variations of this breeding plan could result using the methods of the present invention and these variations are presumed covered provided that the level of CD 151 in a sample of cellular material is determined and this level of CD 151 influences the breeding strategy.
The present invention also provides a method of ascertaining the susceptibility to PRRS infection in animals. Such a method would include the steps of obtaining a sample of cellular material such as tissue or fluid, performing a CD 151 assay on this sample, and using the results of this assay as a measure of the animal""s susceptibility to PRRS infection. The cellular material chosen for the assay can be any cellular material including any fluid or tissue or semi-purified or purified cellular preparation which contains detectible levels of CD 151. For example, blood, blood platelets, germplasm, sperm cells, ova, and semen all contain detectible levels of CD 151. One preferred assay would include the steps of extracting the RNA from the sample and then performing RT-PCR on the extracted RNA. Preferably, the results would provide a quantified amount of detected CD 151. This quantified amount (or level) could then be compared to a known standard for CD 151 levels, thereby indicating the susceptibility to PRRS infection for the animal. Still more preferably, the cellular material sample would be derived from a known origin and the known standard would represent the known standard for cellular material of the same origin.
The present invention further provides a method for determining if an animal is resistant to PRRSV infection based on the presence or absence of CD 151 in cellular material. If CD 151 is absent, the animals will typically be immune to PRRSV infection. If CD 151 is present the animals will typically be susceptible to PRRSV infection to varying degrees based on the actual CD 151 levels. In this manner, PRRSV infection resistance in animals can be classified based on the detected level of CD 151 in any sample of cellular material. Such a method would include the steps of obtaining a sample of cellular material from the tested animal, performing an assay on the sample to find the CD 151 level of that sample, comparing the CD 151 level of the tested animal with a known scale of CD 151 levels wherein the known scale corresponds to a specific degree of PRRSV infection resistance, and finally classifying the tested animal""s PRRSV infection based on the comparison with the known scale. Preferably, the sample of cellular material will be of a known origin and the CD 151 scale will represent the scale for cellular material from the same origin in other animals. Such a classification may result in a percentile ranking of PRRSV infection resistance in the animal.
In another aspect of the present invention, in vitro diagnostic tests for detection of the wild type virus as well as antibodies to the virus are developed. Such test generally include the steps of obtaining a sample of cellular material from an animal, performing a diagnostic test designed to detect either the antibodies to the virus or the virus itself in a recombinant cell line, and using the results of the testing to confirm the diagnosis of PRRS in an individual swine or in a swine herd. Preferred diagnostic tests include virus isolation assays and immunodiagnostic assays such as ELISA, indirect fluorescent antibody tests, and indirect immunoperoxidase tests. Preferably, the recombinant cell line used will permit greater replication of the virus making the test more sensitive to PRRSV infection or antibody presence.
In another aspect of the present invention, higher titer PRRSV vaccines could be developed in CD 151 transformed cell lines to aid in the immunization of swine herds. In a related aspect of the present invention, other non-simian cell lines can be transformed with CD 151 and used to propagate high titer PRRSV vaccine stocks. This has become more of an issue as xenotransplantation (especially in swine) becomes more developed. Use of vaccines made in simian cells may transmit the simian viruses to swine, and hence, organs used for xenotransplantation may also be susceptible to some of these primate viruses. Thus, it will be much safer to avoid using the primate or monkey-kidney cell lines for PRRS vaccines and thereby eliminate the risk of subsequent primate virus introduction into the human transplant recipient population. For cell lines which are already susceptible to PRRS infection, transformation resulting in even higher production of CD 151 can be accomplished using methods of the present invention.
Another related aspect of the present invention will be that the high titer vaccines propagated in non-simian cell lines can be used for other applications such as development of diagnostic tests for detection of antibodies against PRRS, such as an ELISA assay.
Another related aspect of the present invention is the development and use of a plasmid or vector capable of transforming cell lines and enhancing their susceptibility to PRRSV infection. A particularly preferred plasmid in this respect has been given the designation pKSU (Genbank Accession Number AF 275666), and contains the sequence which is provided herein as SEQ ID NO. 1 and is also described in FIG. 1. Preferably, sequences having at least about 91% sequence homology or 93% sequence identity with SEQ ID NO. 1 are embraced by the present invention, whether the sequence appears as an isolated sequence, in a plasmid, or in another suitable vector. More preferably, such sequences will have at least about 95% sequence homology or 97% sequence identity and still more preferably at least about 98% sequence homology or 99% sequence identity with SEQ ID NO. 1. It is believed that this sequence represents the first reported isolated simian CD 151 sequence. Of course, the corresponding amino acid sequences for this and similar sequences are also presumed covered by the present invention as their determination requires no more than routine skill in the art.
Similarly, the present invention provides a method for incorporating CD 151 coding sequences directly in the genome of an animal. This method generally includes the step of integrating the sequence of interest (e.g. CD 151) into the chromosome using a vector designed for such an insertion. In other words, the sequence of interest is incorporated into a retro-viral vector and this retro-viral vector is used to integrate the sequence directly into a chromosome in the genome.
Accordingly, methods for preparing PRRSV vaccine stock are provided by the present invention. In general, to prepare PRRSV vaccine stock, a cell line is provided and then transformed with CD 151. This cell line can be resistant to PRRSV infection, or can be susceptible to PRRSV infection prior to transformation. The resultant transformed cell line is then infected with PRRSV and caused to produce PRRSV progeny for use in the vaccine stock. As noted above, the cell line is preferably of non-simian origin. In this manner, cell lines which are previously not susceptible to PRRSV infection are rendered susceptible after transformation with CD 151 and cell lines which were previously susceptible to PRRSV infection produce much greater numbers of progeny virus. Preferably, the transforming step includes stable transfection with a plasmid containing a CD 151 DNA sequence. The CD 151 sequence can be derived from any animal which has CD 151. Preferably the CD 151 is porcine or simian CD 151. More preferably, the CD 151 DNA sequence has at least about 91% sequence homology with SEQ ID NO. 1. Still more preferably, the CD 151 DNA sequence has at least about 95% and still more preferably at least about 98% sequence homology with SEQ ID NO. 1. The resultant vaccine stock is of higher titer than was previously obtainable prior to transformation. In some cases, this vaccine stock""s titer is about 100 fold higher than was previously possible.
In another aspect of the present invention, polymorphisms in CD 151 sequences are detected and identified using RT-PCR followed by sequencing or a specifically designed test. These different polymorphisms are then analyzed to determine their effects on PRRSV susceptibility. Moreover, the level of CD 151 may be affected by regulatory sequences, such as promoters of the gene. The regulatory sequences are then analyzed to know tissue specific differences and differences among individual piglets.
Still another aspect of the present invention is the development and use of anti-REPs by the Northwestern strategy. Anti-REPs will be an important addition to the arsenal of drugs against emerging viruses. It is now believed that novel emerging viruses will all be RNA and most likely will emerge in wildlife populations due to encroachment of humans to wildlife habitats. These anti-REPs are extremely useful because they provide a readily available strategy that can be used for viral diseases that may emerge in animals (both domesticated and wild) and humans in future. It is important to understand the identification and mechanisms of RNA-binding proteins such as CD 151 for PRRSV and discovery of the compounds belonging to the category of anti-REPs. One potential method for blocking the entry of PRRSV into cells includes the step of blocking PRRSV viral RNA from interaction with CD 151. This blocking is preferably accomplished by contacting the cells with an anti-viral compound, preferably an anti-REP compound. These anti-viral compounds will be designed to occupy binding sites on CD 151, thereby blocking this viral RNA-CD 151 interaction. Preferably, these anti-viral compounds will have a greater affinity as well as a greater avidity for these binding sites than the PRRSV viral-RNA and should be producible in a high throughput manner.
Another aspect of the present invention is the ability of CD 151 to bind to RNA and this is the first report of a tetraspan molecule having RNA-binding activity. This discovery can be used to develop a novel class of compounds that prevents PRRSV or viral RNA-binding to this tetraspan and thus being released into the cell cytosol from the endosome. Combinatorial chemistry and screening by a high throughput screening system can be used to find lead compounds to treat PRRSV and potent anti-REP compounds can be found by a modified Northwestern assay.
PRRSV is a positive sense RNA virus causing serious economic losses in swine. Previous studies have shown that 3xe2x80x2UTR RNA of the arteriviruses plays an important role in the replication of the virus through the interaction with the cellular proteins. A cDNA library of MARC cells was constructed in the xcex ZAP Express vector and the library was screened with positive sense 3xe2x80x2UTR ([xcex1-32P] UTP) RNA of PRRSV. A RNA binding clone with an insert of 1.4 kb was found, and, after sequencing, it exhibited homology to CD 151, a transmembrane protein belonging to the tetraspan family of proteins. The MARC and BHK-21 cells were transfected with the CD 151 plasmid, and the fusion protein was immunoprecipitated with anti-Lac Z and anti-CD 151 antibodies. On Northwestern blotting, the precipitated 29 kD protein interacted with the radiolabelled 3 xe2x80x2UTR. The precise function of CD 151 is not known, but it has been shown to play roles in cell-cell adhesion, regulation of vascular permeability, and transmembrane signaling. The BHK-21 cell line lacks CD 151 and is not susceptible to PRRSV infection, but when the BHK-21 cell line was transfected with the CD 151 plasmid, it became susceptible to PRRSV infection. Similarly, the non-simian cell lines can be transformed with CD 151 and used for PRRSV propigation to high titer.
In order to identify cellular proteins that bind to 3xe2x80x2UTR of PRRS virus, RNA ligand screening of a MARC cell expression library was performed. Moreover, this is the first report of the RNA binding property of a tetraspan molecule, Platelet Endothelial Tetraspan Antigen-3 (PETA-3), also designated as CD 151, and its role in PRRS virus infection. It is known that viruses bind to receptors (protein-protein interaction) and get into the cell in endosome. Under low pH conditions, the virus undergoes structural disorganization and releases viral RNA into the endosome. However, the entry of viral RNA in endosome into cell cytoplasm is not known. The present invention demonstrates that RNA binding proteins, such as CD 151, help PRRSV to enter into cell cytoplasm for further replication of the virus (FIG. 9).